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Cyanobacteria have great potential in CO2-based bio-manufacturing and synthetic biological studies. The filamentous cyanobacterium, Leptolyngbya sp. strain BL0902, is comparable to Arthrospira (Spirulina) platensis in commercial-scale cultivation while proving to be more genetically tractable. Here, we report the analyses of the whole genome sequence, gene inactivation/overexpression in the chromosome and deletion of non-essential chromosomal regions in this strain. The genetic manipulations were performed via homologous double recombination using either an antibiotic resistance marker or the CRISPR/Cpf1 editing system for positive selection. A desD-overexpressing strain produced γ-linolenic acid in an open raceway photobioreactor with the productivity of 0.36 g·m-2·d-1. Deletion mutants of predicted patX and hetR, two genes with opposite effects on cell differentiation in heterocyst-forming species, were used to demonstrate an analysis of the relationship between regulatory genes in the non-heterocystous species. Furthermore, a 50.8-kb chromosomal region was successfully deleted in BL0902 with the Cpf1 system. These results supported that BL0902 can be developed into a stable photosynthetic cell factory for synthesizing high value-added products, or used as a model strain for investigating the functions of genes that are unique to filamentous cyanobacteria, and could be systematically modified into a genome-streamlined chassis for synthetic biological purposes.

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Trichodesmium are filamentous cyanobacteria of key interest due to their ability to fix carbon and nitrogen within an oligotrophic marine environment. Their blooms consist of a dynamic assemblage of subpopulations and colony morphologies that are hypothesized to occupy unique niches. Here, we assessed the poorly studied diversity of Trichodesmium in the Red Sea, based on metagenome-assembled genomes (MAGs) and hetR gene-based phylotyping. We assembled four non-redundant MAGs from morphologically distinct Trichodesmium colonies (tufts, dense and thin puffs). Trichodesmium thiebautii (puffs) and Trichodesmium erythraeum (tufts) were the dominant species within these morphotypes. While subspecies diversity is present for both T. thiebautii and T. erythraeum, a single T. thiebautii genotype comprised both thin and dense puff morphotypes, and we hypothesize that this phenotypic variation is likely attributed to gene regulation. Additionally, we found the rare non-diazotrophic clade IV and V genotypes, related to Trichodesmium nobis and Trichodesmium miru, respectively that likely occurred as single filaments. The hetR gene phylogeny further indicated that the genotype in clade IV could represent the species Trichodesmium contortum. Importantly, we show the presence of hetR paralogs in Trichodesmium, where two copies of the hetR gene were present within T. thiebautii genomes. This may lead to the overestimation of Trichodesmium diversity as one of the copies misidentified T. thiebautii as Trichodesmium aureum. Taken together, our results highlight the importance of re-assessing Trichodesmium taxonomy while showing the ability of genomics to capture the complex diversity and distribution of Trichodesmium populations.

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Differentiation commitment is one of the most complex mechanisms to study in biological science. One of the model systems used for understanding differentiation complexity is heterocyst development in cyanobacteria. Cyanobacteria have the capability of biological nitrogen fixation due to highly differentiated heterocyst cells. Once the nitrogen deficiency signal is perceived by the cyanobacteria, few of its vegetative cells commit toward the development of heterocyst. Heterocyst provides a microoxic environment that is essential for the nitrogenase complex to fix the atmospheric dinitrogen. The entire process of development of heterocyst can be divided into different steps, such as (a) sensing signal and differentiation induction, (b) positional (pattern) determination of heterocyst in the filament, (c) formation of extracellular thick heterocyst-specific layers, and (d) assembly of nitrogen-fixing machinery. Many of the key regulators that are essential for heterocyst formation in these different steps have been identified. Recently, the role of small RNA and interruption DNA elements that influence the heterocyst formation and function has also been identified. In this review article, we have outlined the current understanding of the entire molecular circuit of heterocyst development in a simplistic way. This article focuses on explaining key concepts related to heterocyst development and discusses recent discoveries in this line.

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Cyanobacteria are a morphologically and physiologically diverse group of bacteria, which contains unicellular and multicellular filamentous strains. Some filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, form a differentiated cell called a heterocyst. The heterocyst is a specialized cell for nitrogen fixation and is differentiated from a vegetative cell in response to depletion of combined nitrogen in the medium. In Anabaena PCC 7120, it has been demonstrated that hetR, which encodes a transcriptional regulator, is necessary and sufficient for heterocyst differentiation. However, comprehensive genomic analysis of cyanobacteria has shown that hetR is present in non-heterocyst-forming cyanobacteria. Almost all filamentous cyanobacteria have hetR, but unicellular cyanobacteria do not. In this study, we conducted genetic and biochemical analyses of hetR (NIES39_C03480) of the non-heterocyst-forming cyanobacterium Arthrospira platensis NIES-39. HetR of A. platensis was able to complement the hetR mutation in Anabena PCC 7120 and recognized the same DNA sequence as Anabaena HetR. A search of the A. platensis genome revealed the HetR-recognition sequence within the promoter region of NIES39_O04230, which encodes a protein of unknown function. Expression from the NIES39_O04230 promoter could be suppressed by HetR in Anabaena PCC 7120. These data support the conclusion that NIES39_O04230 is regulated by HetR in A. platensis NIES-39.

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The cyanobiont Trichormus azollae lives symbiotically within fronds of the genus Azolla, and assimilates atmospheric nitrogen upon N-limitation, which earmarks this symbiosis to be a valuable biofertilizer in rice cultivation, among many other benefits that also include carbon sequestration. Therefore, studying the regulation of nitrogen fixation in Trichormus azollae is of great importance and benefit, especially the two topmost rungs of regulation, the NtcA and HetR transcription factors that are able to regulate the expression of myriads of downstream genes. Bioinformatics tools were used to zoom in on the NtcA and HetR transcription factors from Trichormus azollae to elaborate on what makes this particular cyanobiont different from other symbiotic as well as more distinct counterparts, in their commitment to nitrogen fixation. The utility of Azolla plants in tropical agriculture in particular merits the "top down N-regulation" by cyanobiont as a significant niche area of study, to make sense of superior N-fixing capabilities. The Trichormus azollae NtcA sequence was found as a phylogenetic outlier to horizontally infecting cyanobionts, which points to a distinct identity compared to symbiotic counterparts. There were borderline (60%-70%) levels of acceptable bootstrap support for the phylogenetic position of the Azolla cyanobiont's NtcA protein compared to other cyanobionts. Furthermore, the NtcA global nitrogen regulator in the Azolla cyanobiont has an extra cysteine at position 128, in addition to two other more conspicuous cysteines (positions, 157 and 164). A simulated homology model of the NtcA protein from Trichormus azollae, points to a single unique cysteine (Cysteine-128) as a key residue at the center of a lengthy C-helix, which forms a coiled-coil interface, through likely disulfide bond formation. Three cysteine (Cysteines: 128, 157, 164) architecture is exclusively found in Trichormus azollae and is absent in other cyanobacteria. A separate proline to alanine mutation in position 97-again exclusive to Trichormus azollae-appears to influence the flexibility of effector binding domain (EBD) to 2-oxoglutarate. The Trichormus azollae HetR sequence was found outside of horizontally-infecting cyanobiont sequences that formed a common clade, with the exception of the cyanobiont from the genus Cycas that formed one line of descent with the Trichormus azollae counterpart. Five (out of 6) serines predicted to be phosphorylated in the Trichormus azollae HetR sequence, are conserved in the Nostoc punctiforme counterpart, showcasing that phosphorylation is likley conserved in both vertically-transmitted and horizontally-acquired cyanobionts. A key Serine-127, within a conserved motif TSLTS, although conserved in heterocystous subsection IV and V cyanobacteria, are mutated in subsection III cyanobacteria that form trichomes but are unable to form heterocysts. I conclude that the NtcA protein from Trichormus azollae to be strategically divergent at specific amino acids that gives it an advantage in function as a 2-oxoglutarate-mediated transcription factor. The Trichormus azollae HetR transcription factor appears to possess parallel functionality to horizontally acquired counterparts. Especially Cysteine-128 in the NtcA transcription factor of the Azolla cyanobiont is an interesting proposition for future structure-function studies.

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In the filamentous cyanobacterium, Anabaena sp. PCC 7120, single heterocysts differentiate at semi-regular intervals in response to nitrogen stepdown. HetR is a principal regulator of heterocyst differentiation, and hetP and hetZ are two genes that are regulated directly by HetR. In a hetR mutant generated from the IHB (Institute of Hydrobiology) substrain of PCC 7120, heterocyst formation can be restored by moderate expression of hetZ and hetP. The resulting heterocysts are located at terminal positions. We used a tandem promoter, PrbcLPpetE, to express hetZ and hetP strongly in the hetR mutant. Co-expression of hetZ and hetP enabled the hetR mutant to form multiple contiguous heterocysts at both terminal and intercalary positions. Expression of hetZ, alone resulted in terminally located heterocysts, whereas expression of hetP, alone produced enlarged cells in strings. In the absence of HetR, formation of heterocysts was insensitive to the peptide inhibitor, RGSGR.

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Phylogenetic relationships among heterocytous genera (the Nostocales order) have been profoundly modified since the use of polyphasic approaches that include molecular data. There is nonetheless still ample scope for improving phylogenetic delineations of genera with broad ecological distributions, particularly by integrating specimens from specific or up-to-now poorly sampled habitats. In this context, we studied 36 new isolates belonging to Chrysosporum, Dolichospermum, Anabaena, Anabaenopsis, and Cylindrospermopsis from freshwater ecosystems of Burkina-Faso, Senegal, and Mayotte Island. Studying strains from these habitats is of particular interest as we suspected different range of salt variations during underwent periods of drought in small ponds and lakes. Such salt variation may cause different adaptation to salinity. We then undertook a polyphasic approach, combining molecular phylogenies, morphological analyses, and physiological measurements of tolerance to salinity. Molecular phylogenies of 117 Nostocales sequences showed that the 36 studied strains were distributed in seven lineages: Dolichospermum, Chrysosporum, Cylindrospermopsis/Raphidiopsis, Anabaenopsis, Anabaena sphaerica var tenuis/Sphaerospermopsis, and two independent Anabaena sphaerica lineages. Physiological data were congruent with molecular results supporting the separation into seven lineages. In an evolutionary context, salinity tolerance can be used as an integrative marker to reinforce the delineation of some cyanobacterial lineages. The history of this physiological trait contributes to a better understanding of processes leading to the divergence of cyanobacteria. In this study, most of the cyanobacterial strains isolated from freshwater environments were salt-tolerant, thus suggesting this trait constituted an ancestral trait of the heterocytous cyanobacteria and that it was probably lost two times secondarily and independently in the ancestor of Dolichospermum and of Cylindrospermopsis.

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The filamentous, colonial cyanobacterium Trichodesmium has six well-described species, but many more names. Traditional classification was based on field samples using morphological characteristics such as cell width and length, gas vesicle distribution, and colony morphology. We used the Woods Hole Trichodesmium culture collection to identify 21 cultured strains to species using cell morphology; phycobiliprotein absorption spectra; and sequences of the 16S rRNA gene, the 16S-23S internal transcribed spacer (ITS), and the heterocyst differentiation gene hetR. We compared our results to previous studies of field specimens and found similar clades, though not all phylogenetic groups were represented in culture. Our culture collection represented two of the four major clades of Trichodesmium: clade I, made up of Trichodesmium thiebautii Gomont, Trichodesmium tenue Wille, Katagnymene spiralis Lemmerm., and Trichodesmium hildebrandtii Gomont; and clade III, consisting of Trichodesmium erythraeum Ehrenb. and Trichodesmium contortum Wille. These clades were genetically coherent with similar phycobiliprotein composition, but morphologically diverse. In the continual revision of cyanobacterial taxonomy, genetic and biochemical information is useful and informative complements to morphology for the development of a functional classification scheme.